Meeting 11

Meeting 11 - Giardia we're talking about the ability to...

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Unformatted text preview: Giardia we're talking about the ability to create encystation excystation in the laboratory. It gives an observation of a switching of the surface protein. The virulence coating. It switches encystation and excystation. They had started off a populationtion of trophozoites stained with an antibody with one type of VSP TSA 417 and it was 100% positive with staining. Then they took them and cuased them undergo encystatio nwith high pH and excystation with low pH and trypsin. You see that very few of the parasites now stain with this antibody. They used an alternative way to look at the protein in the population. This is a Western blot experiment I've already talked about. The protein is disappearin gsometime after excystation. In C 5- 48 is another VSP that is coming up after the intial VSP was leaving. They concluded that during encystation and excystation, you turn off one VSP and turn another one on. How to control? Some sort of selective intraecllular transport to the surface. You switch it because you no longer put it on the surface. Does IFA address this? They showed it's not on the surface due to the TSA417. If the antibody could get in the cell, it would show you that ther's simply no protein. It dpepedends. Would the immunoblot tell you? That there's selective intracellular transport? What's put on the immunoblot? total lysate. So no, it would not tell you if there was intracellular or not. Protein stability, they wouldn't know why it's there or not. Production? Selective translation? Youc an't tell from those types of analsysis. You would have to look at the RNA. Not just the protein. The 1st set of experiemtns say if the protein is there or not but not why it's there. Is the protein no longer there because RNA isn't made? That's what they did next. They used Northern blot analysis. RTPCR is a little bit overkill. It's much more sensitive. It can be somewhat more quantitative. These would allow you to examine steady-state RNA lelvels but it won't tell you if the control is at the transcription level. Those are the type of things that allow you to look at the transcriptional level. Runoff gel. You'd do it before anad after encystation andbefore and after excystation. That matrix can be reacted with antisense of the RNA as a probe to detect if the RNA is there. Right isstarting and left is ending . Very dramatic drop between after encystatioin and after encystation. By 90 minutes after you start the excystation, you see you can't deetect any of the RNA. These bottom is loading, the middle probe for mnay VSPs. You see other VSPs coming on as the top is turning off. It says then, the RNA is actually turned off somewhere ner the stage of excystation. In the case of the trypanosome, is how to get the VSG off the surface. For the VSP, no one's really addressed if they are degraded. There is probably some way to get ride of it. It indicates the RNa disappers during excystation. We know that it's not transcriptional regulation. We were told that VSG is turned on an off by the transcriptional regulation. Either turn on or off promoter. VSP appears to be controlled post transcriptinally. They think that this is regulated by RNAi. It's very recent. It indicates the way you select for a single VSP to be expressed to be put on the surface is to use a RNAi degradation to degrade all the other VSPs except fot he one VSP that will be used. It shoulds like a very wasteful way to be done. They do not have polycistronic transcription. The only polycistronic euk cell are kinetoplastids. All the genes in all the other bugs appear to be monocistronic. Conclude fromthis that they noticed during encystation and excystation they turn off the old VSP and turn on the new VSP. This is switching during differentiation. If you tahink of encystation and excystation as a life cycle, you switch upon completeiono f a life cycle when transmitting from one host to another. It keeps the population infected with giardia. People can reinfect themselves. Even if you raise an Ab response, if you pass cyst out of your body during encystation and excystation and then if you injest the new cyst you won't have any antibody against the VSP because it's a new VSP. It lives in a lot of reservoir hosts. Instead of topping out where every one has seen the same VSP, if you change the VSP, you d can establish a new infection. It's an attractive model, there are also other stimuli to induce switching but it is one of the way. For Giardia, it's not known how random they pick a new VSP. In the lab, it is ABSOLUTELY not random for trypanosomes, dont' know if that's just laboratory artifacts. For giarida, not much is known. Antigenic variation was discovered in 1978, they still don't know. In giardia, any mechanistic studies only started 10 years ago. Today's lecture! Entamoeba, this is a global death chart around the world. you see that we have an enormous percentuage to repiratory, diarrhea, and tuberculosis. There's also specific ones. There's over 3 million deaths caused by diarrheal infections. Entamoeba histolytica, it's the 3rd leading death due to parasitic diseases. Schistosoma is this worm parasite. Entamoeba could be considered a good parasite because it doesn't kill the host. You could define a parasite a good parasite if it doesn't kill. Commenusal doesn't cause disease, but a parasite does cause disease but is not lethal. Here's another chart, this differentiates between dcuases of death worldwide. It differentiates with over the age of 5. You can see with diarrheal, the bulk majority of those killed are below the age of 5. Whereas you have a differe profile for AIDS or TB. This just shows you a schart of percentages under the age of 5 that are dying in these selected countries due to a preventable disease. The black bit is the diarreal. Goes hand in hand with malnutrition. This gives you a sense of where you encounter these problems. Here's a village in bangladesh, their daily activities bring them in contact with contaminated water. The cyst is the infective form. They don't even realize that by drinking this water is giving them infection. One in ten children in Banglaadesh die before their 5th birthday. These are preventable diseases. Amoebiasis. Another way of viewing life cycle. The cyst can get injested, they go to small intestines and under go excystation and establishes infection in the mucosal surface in the colon. In some people, it can breach the mucosa go throughout the body to the brain and the liver. This is the most common place, the liver. They don't ever transmit to another host, just the ones that are in the colon that under go encystation. It's an enteric amoeba that lysises tissues, you're histolytic. It extends pseudopodia. How does Giardia move around? With flagella,this is an amoeba. It sends cytockeltal structures out and it moves out forward. It also engulfs and digests bacteria but also RBCs and host cells. It 's cytolytic to the tissue of the colon. It's an entamoeba phagocytosing a red cell. You see it's phyagocytic capacity and how big it is. Here's a staining fo the trophozoite. You see these stained dotrks, the RBCs within the entamoeba. The Nucleus is single. you also see this refactile vacuole. It's rather big, lysozomal, there are multiple and they're huge. It probably depends on how digestive it is. another characteristic is that you can see by EM is that arere are all these rybonucleo particules, these little helixes. They're not ribosomes or splicsosomes, tbut they're akin that they have ribo nucleo particles. Thisisi a cyst. In the frame, you see a single nuclei, but these cysts actually have 8. during excystation, they form 8.???? This is one of the main ways of diagnosis. You look at the feces and look for it. This isn't very good since there are way a lot of organisms that look like it. So instead, there are good PCR based assays. You look for the two criteria for an assay is that it's sensitive and specificity. It's very sensitive an dvery specifict. You can say for sure you see it. this is the ideal way, but this is not practical or you don' thave the facilities in case of contamination. There's an ELISA antibody based assay but the sensitivity is not so great. There's also Microscopy. The sensitivity isn't that bad, but the specificyt is really bad. You just can't distinguish whether or not is pathogenic or not. There are a lot of pathogenic amoeba and there's a concern with overtreating people for diarrhea. Overtreatment is never desired. This parasite also excretes toxic factors. It can cause ulcers in the large intesteincs. This just shows you a scaled down cartoon. You get these trophozoites growing int he entestine. If they get to the liver, you get these horrible abscesse. These are the ulcers. This showsentamoeba in the tissues of the animal. Here is the worst, liver abscess. It's an enormous amount. not all strains of entamoeba will breach the mucosa, there's evidence that it also depends on the genetic even with the same strain in some places. The exact human genetic studies have not been done, but it's due to the genetic background of the host. Once you have an amoebic abscess, if it's not treated, but it's pretty much fatal. There are really goo ddrugs to trea the abscess but they're simply not detected. Virulence factors = any molecule of any sort produced by the parasite that help establish infection, cause disease, need to transmission, evade host immune defenses, etc and more. Anything that allows the parasite to be established and maintain is a virulence factor. Adherence factors are very important for extracellular parasites like entamoeba. Also important for intracellular since they need to bind to the host cell. Invasion factors are really important for intracellulr parasites, and without them you can't go in the cell. Endotoxins, such as capsules like LPS that often coat the surface. They're not actively secreted necessarily but they are released. There are exotoxins that re truly secreted. by an active mechanism. Then there are siderophores, very well studied in bacteria, substances that extract iron from host. We're going to focus on the best studied VF of the entamoeba. GalNAc lectin, cysteine proteinases, amoebapore (specialized proteins involved in lysis of target cells), etc. The skeleton could be called a Vf since it's involved in motility etc. GalNAC lectin is involved in adherence. it's the bold little rectangles to the left. Cysteine roteases to the right and amoebapore on the bottom. This shows amoeba cell that bind 5 different host cells. It le to that human cells require adherence for the amoeba cell to bind and carbohydrates. It's an ability of a parasite to specifically bind to carbohydrate structures of the host. Called the Gal/GalNac Lectin. How was it discovered? Early evidence is that in vitro cell binding assay sing colonic epthelia cells, if you add 50mM glaactose or gal nac, it blocks the binding of the parasite to the host cells. That data alone gave the clue that if you add excess amoutn of the sugar, the parasite binds the free sugar in the assay, there are no binding sites for the parasite to bind to the host cell. They take host cells that are mutant in glycolsylation enzymes, you find that the parasite will not kill these mutant or adhere to theses mutant cell lines. If you mutate O and N linked, you almost negate the adherence. It led to further ideas of sugar on the surface of the host cell playing a role. Petri assayed ... whatever binds to the galactose is a surface molecule on the parasite. He made an affinity chromatogrphy column. He took a mtraix and hooked galactose to this column and took the parasites and lysed them, solubilized the membranes with determgent and then put the gamesh over the galactose column. You wouldn't do it now, but the concept is the same. He had to do it was label the parasites prior to lysing them with S35, all the proeins would be labeled. He started washing this column, he saw the bulk of the proteins didn't bind. Almost all of it washed off, then he started collecting fraction. There was almost no radiactivity, which means that he's washed off all the proteins. Then he bumped it off, eluting with galactose, then it will equilibrium, the proteins will pop off and bind to the free galactose. Sure enough that is what happens. He saw a labeld protein. He rusn it on a gell and youc an see that it's incredibily clean, he saw two specific bands. He got the peptide sequenced. you would sequence via mass spect and design primers, and then PCR and pick up the gene. Another way that nobody does anyomre was produce antibodies and screen a cDNA expression library. Once you get the protein, youc an get the gene and vice versa. He translates it and this is what he got. He got surface ectin. The two proteins are bound together by disulfide linkages. There's also a carbohydrate recognition domain. It also actually sits int he membrane. This is now known from a lot of studies. The 170kD piece actually goes through the transmembrane and then there's a signaling tail. It binds to host celland then the signals phosphorylation and kinases. Signaling pathways occur through Cterminal tail. They call it the heavy and light subunit. It's linked through a lipid anchor to the membrane. They're bonded together by disulfide bonds. There are proteolytic sites. The host can cleave it off. Once you hvave the gene, you can overexressyou can also silence the gene ansee what efects on pathogenesis. Overexpression cartoon. You could add back the lectin via transfection and that will send the proteins to the surface, you coan inactivate it, bu i you have active you will get greater binding. They use this technology to introduce dominant negative that is mutated such as mutate the signaling tail. KEY is to find the molecule to study with. This indicates that it mediates aadherence withe pithelial, muzin, and immune host cells. This just lists a number of the different things this GAl GalNAc lectin can do through these asssays. It is known now toa dherence for sure and it induces apoptosis of host cells. You can decrease the level of the light chain of mRNA band those then are correclated with reduced ability to ill host cell. it seems to medate colon uptake. you can downregulate if you have a lot of bcteria. It induces an antibody response in infected children and plays a role in giving them immunity. We're now trying to look here to get a vaccine....
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